Studying the secondary structure of proteins leads to an understanding of the components that make up a whole protein, and such an understanding of the structure of the whole protein is often vital to understanding its digestive behaviour and nutritive value in animals. The main protein secondary structures are the α-helix and β-sheet. The percentage of these two structures in protein secondary structures influences protein nutritive value, quality and digestive behaviour. A high percentage of β-sheet structure may partly cause a low access to gastrointestinal digestive enzymes, which results in a low protein value. The objectives of the present study were to use advanced synchrotron-based Fourier transform IR (S-FTIR) microspectroscopy as a new approach to reveal the molecular chemistry of the protein secondary structures of feed tissues affected by heat-processing within intact tissue at a cellular level, and to quantify protein secondary structures using multicomponent peak modelling Gaussian and Lorentzian methods, in relation to protein digestive behaviours and nutritive value in the rumen, which was determined using the Cornell Net Carbohydrate Protein System. The synchrotron-based molecular chemistry research experiment was performed at the National Synchrotron Light Source at Brookhaven National Laboratory, US Department of Energy. The results showed that, with S-FTIR microspectroscopy, the molecular chemistry, ultrastructural chemical make-up and nutritive characteristics could be revealed at a high ultraspatial resolution (∼10 μm). S-FTIR microspectroscopy revealed that the secondary structure of protein differed between raw and roasted golden flaxseeds in terms of the percentages and ratio of α-helixes and β-sheets in the mid-IR range at the cellular level. By using multicomponent peak modelling, the results show that the roasting reduced (P<0·05) the percentage of α-helixes (from 47·1 % to 36·1 %: S-FTIR absorption intensity), increased the percentage of β-sheets (from 37·2 % to 49·8 %: S-FTIR absorption intensity) and reduced the α-helix to β-sheet ratio (from 0·3 to 0·7) in the golden flaxseeds, which indicated a negative effect of the roasting on protein values, utilisation and bioavailability. These results were proved by the Cornell Net Carbohydrate Protein System in situ animal trial, which also revealed that roasting increased the amount of protein bound to lignin, and well as of the Maillard reaction protein (both of which are poorly used by ruminants), and increased the level of indigestible and undegradable protein in ruminants. The present results demonstrate the potential of highly spatially resolved synchrotron-based infrared microspectroscopy to locate ‘pure’ protein in feed tissues, and reveal protein secondary structures and digestive behaviour, making a significant step forward in and an important contribution to protein nutritional research. Further study is needed to determine the sensitivities of protein secondary structures to various heat-processing conditions, and to quantify the relationship between protein secondary structures and the nutrient availability and digestive behaviour of various protein sources. Information from the present study arising from the synchrotron-based IR probing of the protein secondary structures of protein sources at the cellular level will be valuable as a guide to maintaining protein quality and predicting digestive behaviours.

Synchrotron radiation-based Fourier transform IR (SR-FTIR) microspectroscopy has been developed as a rapid, direct, non-destructive and bioanalytical technique. This technique, taking advantage of synchrotron light brightness and a small effective source size, is capable of exploring the molecular chemistry within the microstructures of a biological tissue without the destruction of inherent structures at ultraspatial resolutions within cellular dimensions. This is in contrast to traditional ‘wet’ chemical methods, which, during processing for analysis, often result in the destruction of the intrinsic structures of feeds. To date there has been very little application of this technique to the study of feed materials in relation to animal nutrient utilisation. The present article reviews four applications of the SR-FTIR bioanalytical technique as a novel approach in animal nutrition and feed science research. Application 1 showed that using the SR-FTIR technique, intensities and the distribution of the biological components (such as lignin, protein, lipid, structural and non-structural carbohydrates and their ratios) in the microstructure of plant tissue within cellular dimensions could be imaged. The implication from this study is that we can chemically define the intrinsic feed structure and compare feed tissues according to spectroscopic characteristics, functional groups, spatial distribution and chemical intensity. Application 2 showed that the ultrastructural–chemical makeup and density of yellow- and brown-seeded Brassica rape could be explored. This structural–chemical information could be used for the prediction of rapeseed quality and nutritive value for man and animals and for rapeseed breeding programmes for selecting superior varieties for special purposes. More research is required to define the extent of differences that exist between the yellow- and brown-seeded Brassica rape. Application 3 showed with the SR-FTIR technique that chemical differences in the ultrastructural matrix of endosperm tissue between Harrington (malting-type) and Valier (feed-type) barley in relation to rumen degradation characteristics could be identified. The results indicated that the greater association of the protein matrix with the starch granules in the endosperm tissue of Valier barley may limit the access of ruminal micro-organisms to the starch granules and thus reduce the rate and extent of rumen degradation relative to that of Harrington barley. It is the first time that the microstructural matrix in the endosperm of barley has been revealed by using the SR-FTIR technique, which makes it possible to link feed intrinsic structures to nutrient utilisation and digestive behaviour in ruminants. Application 4 showed with the SR-FTIR technique that the chemical features of various feed protein (amide I) secondary structures (such as feather, wheat, oats and barley) could be quantified. With a multi-component fitting program (Lorentz function), the results showed feather containing about 88% β-sheet and 4% α-helix, barley containing about 17% β-sheet and 71% α-helix; oats containing about 2% β-sheet and 92% α-helix; and wheat containing about 42% β-sheet and 50% α-helix. The relative percentage of the two may influence protein value. A high percentage of β-sheet may reduce the access of gastrointestinal digestive enzymes to the protein structure. Further study is required on feed protein secondary structures in relation to enzyme accessibility and digestibility. In conclusion, the SR-FTIR technique can be used for feed science and animal nutrition research. However, the main disadvantage of this technique is the requirement for a special light source; a synchrotron beam.

The objectives of this study were to reveal protein structures of feed tissues affected by heat processing at a cellular level, using the synchrotron-based Fourier transform infrared microspectroscopy as a novel approach, and quantify protein structure in relation to protein digestive kinetics and nutritive value in the rumen and intestine in dairy cattle. The parameters assessed included 1) protein structure alpha-helix to beta-sheet ratio; 2) protein subfractions profiles; 3) protein degradation kinetics and effective degradability; 4) predicted nutrient supply using the intestinally absorbed protein supply (DVE)/degraded protein balance (OEB) system for dairy cattle. In this study, Vimy flaxseed protein was used as a model feed protein and was autoclave-heated at 120 degrees C for 20, 40, and 60 min in treatments T1, T2, and T3, respectively. The results showed that using the synchrotron-based Fourier transform infrared microspectroscopy revealed and identified the heat-induced protein structure changes. Heating at 120 degrees C for 40 and 60 min increased the protein structure alpha-helix to beta-sheet ratio. There were linear effects of heating time on the ratio. The heating also changed chemical profiles, which showed soluble CP decreased upon heating with concomitant increases in nonprotein nitrogen, neutral, and acid detergent insoluble nitrogen. The protein subfractions with the greatest changes were PB1, which showed a dramatic reduction, and PB2, which showed a dramatic increase, demonstrating a decrease in overall protein degradability. In situ results showed a reduction in rumen-degradable protein and in rumen-degradable dry matter without differences between the treatments. Intestinal digestibility, determined using a 3-step in vitro procedure, showed no changes to rumen undegradable protein. Modeling results showed that heating increased total intestinally absorbable protein (feed DVE value) and decreased degraded protein balance (feed OEB value), but there were no differences between the treatments. There was a linear effect of heating time on the DVE and a cubic effect on the OEB value. Our results showed that heating changed chemical profiles, protein structure alpha-helix to beta-sheet ratio, and protein subfractions; decreased rumen-degradable protein and rumen-degradable dry matter; and increased potential nutrient supply to dairy cattle. The protein structure alpha-helix to beta-sheet ratio had a significant positive correlation with total intestinally absorbed protein supply and negative correlation with degraded protein balance.